Project Summary Membrane proteins are abundant in eukaryotic cells and play important roles in a great many biological processes ranging from cell adhesion and recognition to energy production to signaling cascades. Furthermore, membrane proteins make up about 60% of the targets for currently approved drugs, which underscores their relevance to human disease. Although very high resolution structures of a number of membrane proteins have now been solved, we lack methods that will also allow us to resolve the structures of the membrane lipids that interact with membrane-embedded proteins, peripheral membrane proteins and other ligands. This is in spite of the fact that specific membrane lipids play key regulatory roles in biology. We term these ?functional lipids? because, in addition to their well-known structural roles in membranes, it is becoming increasingly clear that lipids are effector molecules that modulate and/or directly carry out essential biological functions. An atomic-scale understanding of the interactions carried out by functional lipids is an important unmet goal with direct relevance to human health and disease. Thus, although excellent methods now exist for solving the structures of proteins?including membrane proteins?at very high resolution, the field lacks tools necessary to solve the structures of the lipid part of membranes at high resolution. This ambitious project aims to develop an innovative ?toolkit? of high-resolution methods for the scientific community to use in solving the structures of lipids that regulate the biological functions of membranes. Our approach requires synergistic and coordinated efforts throughout: (1) cost-effective, site-specific isotopic labeling of a variety of phospholipids and sterols; (2) assembling labeled lipids into nanoscale lipid bilayer systems together with their biologically relevant ligands; (3) nuclear magnetic resonance (NMR) approaches, principally high-field magic-angle spinning solid-state NMR (SSNMR), to obtain detailed structural information about the lipids interacting with ligands; (4) cutting-edge computational methods employing molecular dynamics (MD) simulations of lipids interacting with ligands in bilayers or bilayer mimetics; and (5) new methods for solving lipid structures by marrying computational NMR and MD approaches to address the unique challenges inherent in interpreting and understanding spectral data obtained from planar bilayers that contain repeating copies of labeled lipids interacting with neighboring lipids in addition to their specific ligands. As our studies progress, we propose to apply this toolkit to exemplary problems in biology, including blood coagulation, antimicrobial peptide action and sterol recognition.